Natural regulatory T cells (CD25+, CD45RBlo) appear to be responsible for controlling autoreactive T cell activity and, thereby, the development of autoimmune diseases. They are present in naive mice and express an activation/memory surface marker phenotype. These cells do not proliferate in response to a variety of stimuli, but are very potent inhibitors of effector T cell proliferation in vitro. Although some progress has been made in understanding their mechanisms of action, little is known about their requirements for development or peripheral activation. Evidence suggests that natural regulatory T cells may undergo selection/activation at both the thymus and peripheral sites, such as the mucosa. The gut, in particular, is a strong candidate for this mucosal site since exposure to antigen through these tissues tends to result in tolerance rather than immunity, and the antigenic load is huge and extremely diverse. We propose that natural regulatory T cell development requires two stages of activation to become fully functional; an induction and an effector stage, both of which may involve the gut mucosa. Using in vitro and in vivo assays, our studies will address three specific aims: (1) to characterize the basic requirements for development of natural regulatory T cells; (2) to determine whether GALT is involved in the induction stage of natural regulatory T cell development; and (3) to determine whether GALT is critical for the secondary activation of natural regulatory T cells in the periphery. These experiments may determine the requirements for regulatory T cell development, and thereby, identify pathways that can be therapeutically manipulated to prevent and/or treat autoimmune diseases.